Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Hemophilia is a genetic disease characterized by a blood clotting deficiency. In hemophilia A (classic hemophilia, Factor VIII deficiency), an X-chromosome-linked genetic defect disrupts the gene encoding Factor VIII, a plasma glycoprotein, which is a key component in the blood clotting cascade. The cDNA sequence encoding human Factor VIII is available at GenBank with Accession No. K01740 (SEQ ID NO: 1). Within SEQ ID NO: 1, nucleic acids 208–7206 encode the full-length wild-type FVIII polypeptide (2332 amino acids, SEQ ID NO: 2) and nucleic acids 151–207 encode a preceding 19-residue signal sequence peptide (SEQ ID NO: 3). Human Factor VIII may be synthesized as a single chain polypeptide, with a predicted molecular weight of 265 kDa. The Factor VIII protein (SEQ ID NO: 2) has six domains, designated from the amino to the carboxy terminus as A1-A2-B-A3-C1-C2 (Wood et al., Nature 312:330 [1984]; Vehar et al., Nature 312:337 [1984]; and Toole et al., Nature 312:342 [1984]). Human Factor VIII is processed within the cell to yield a heterodimer primarily comprised of a heavy chain of 200 kDa containing the A1, A2, and B domains and an 80 kDa light chain containing the A3, C1, and C2 domains (Kaufman et al., J. Biol. Chem., 263:6352–6362 [1988]). Both the single chain polypeptide and the heterodimer circulate in the plasma as inactive precursors (Ganz et al., Eur. J. Biochem., 170:521–528 [1988]). Activation of Factor VIII in plasma is initiated by thrombin cleavage between the A2 and B domains, which releases the B domain and results in a heavy chain consisting of the A1 and A2 domains. The 980 amino acid B domain is deleted in the activated procoagulant form of the protein. Additionally, in the native protein, two polypeptide chains (“a” and “b”), flanking the B domain, are bound to a divalent calcium cation. Hemophilia may result from point mutations, deletions, or mutations resulting in a stop codon (See, Antonarakis et al., Mol. Biol. Med., 4:81 [1987]).
The disease is relatively rare, afflicting approximately one in 10,000 males. Hemophilia in females is extremely rare, although it may occur in female children of an affected father and carrier mother, as well as in females with X-chromosomal abnormalities (e.g., Turner syndrome, X mosaicism, etc.). The severity of each patient's disease is broadly characterized into three groups—“mild,” “moderate,” and “severe,” depending on the severity of the patient's symptoms and circulating Factor VIII levels. While normal levels of Factor VIII range between 50 and 200 ng/mL plasma, mildly affected patients have 6–60% of this value, and moderately affected patients have 1–5% of this value. Severely affected hemophiliacs have less than 1% of normal Factor VIII levels.
While hemophiliacs clearly require clotting factor after surgery or severe trauma, on a daily basis, spontaneous internal bleeding is a greater concern. Hemophiliacs experience spontaneous hemorrhages from early infancy, as well as frequent spontaneous hemarthroses and other hemorrhages requiring clotting factor replacement.
Without effective treatment, chronic hemophilic arthropathy occurs by young adulthood. Severely affected patients are prone to serious hemorrhages that may dissect through tissue planes, ultimately resulting in death due to compromised vital organs.
Clearly a need exists for improved compositions and methods for the treatment of this genetic disorder.